1 Field of Invention
The invention relates to a method and a device for processing a biological sample. Processing within the sense of the present invention comprises binding, washing and eluting biomolecules of the biological sample.
2 Description of Related Art
Processing of biological materials is used, for example, in the field of extracting and/or purifying biomolecules such as nucleic acids or proteins. For example, a widely known method of purifying the biomolecules is based on the steps of making the contents of a biological sample accessible (“lysis”), selectively binding the constituents of the contents of the biological sample to or on a solid support or carrier material (“binding”), eliminating unwanted constituents from the solid support or carrier material (“washing”) and dissolving the desired constituent (“eluting”).
In order to permit a desired absorption or desorption of the biomolecules during the purification of the biomolecules, special filter elements have been developed which are formed, for example, from silica gel, and which are, on the one hand, porous or matrix-like, in order to let a liquid pass through the filter element, and which, on the other hand, have a surface to which the biomolecules bind in a specific or unspecific process. In other purification methods, biomolecules are retained on the filter elements simply due to the size exclusion effect. If a liquid containing a biomolecule, such as a nucleic acid, passes the filter element, the biomolecules or a part thereof remain in the filter element in any case, whereas the rest passes through the filter element. Furthermore, in order to obtain the biomolecule from the filter element, an eluting liquid, e.g. nuclease-free water, is brought onto the filter element for desorbing the biomolecule. In this manner, the desired biomolecule is dissolved from the filter element (eluted) and collected in a vessel. Such filter elements are frequently designed as membranes, which are either disposed in individual vessels having an inlet opening and an outlet opening, or which are disposed in multiwell plates. The filter elements are processed either with centrifuges (“spin format”) or with apparatuses based on vacuum technology. Individual vessels with an inlet opening and an outlet opening which have a membrane and which can be used in a centrifuge are also known as columns, centrifuging columns, filtering vessels, chromatography columns, “columns”, “spin columns” or “single spin columns”.
Generally, the advantages of the centrifuging method compared with the vacuum-based methods include a higher degree of purity, a higher concentration and a lower risk of cross contamination. Generally, the best results for the purification of the biomolecules with regard to quality and concentration can be obtained with the centrifuging columns (single spin columns), which are processed under a field of high gravity (>10,000×g), because a minimum cross contamination and a maximum recovery of the desired substance in eluting liquid from the membrane is possible. One drawback, however, is the labor-intensive manual treatment of the centrifuging columns, which increases the risk of error and the processing time, in particular if different samples are to be treated or processed simultaneously. A higher degree of standardization and automation, as well as a higher throughput can be achieved by using multiwell plate formats.
The company QIAGEN GmbH, from Hilden, Germany, offers a broad spectrum of purification protocols and products required therefor for different biomolecules from a multitude of biological samples based on the basic principle of the “bind-wash-elute” protocol. The product “QIAGEN QIAprep Spin Miniprep Kit”, for example, is commercially available. For this purpose, different filter materials and devices are used, such as are described, for example, in WO 03/040364 or U.S. Pat. No. 6,277,648.
It is thus known to use a vessel for processing a sample that is open towards the top and sealable. A membrane as a filter element is located at the bottom of the vessel. An opening in the form of a port, which is connected, for example, to a tube, is located under the membrane. Liquid can be withdrawn from the vessel via this tube.
For processing, the biological sample is put into the vessel at the top. Then, the solution which is respectively provided is added, that is, for example, first a lysis buffer in order to disrupt the sample. When the sample has been disrupted, the lysis buffer is withdrawn from the vessel in a downward direction. The lysate contains chemical/physical conditions causing the nucleic acid to bind to the carrier material. The disrupted sample (lysate) is then washed and eluted.
After the nucleic acid of the disrupted sample was bound or adsorbed to the membrane, washing buffers are put into the vessel from the top and withdrawn in a downward direction from the vessel or centrifuged. The washing buffer maintains this binding while simultaneously washing unwanted cellular constituents from the membrane.
As a rule, washing buffers contain ethanol. Ethanol causes nucleic acid to bind to a membrane acting as a filter element. A membrane acting as a filter has a certain dead volume of, for example, 40 μl. It can therefore not be avoided that a corresponding amount of ethanol always remains in the filter. On the one hand, ethanol undesirably contributes to binding. On the other hand, ethanol can also corrupt the results of a later analysis. In order to be able to remove the nucleic acid from the vessel in a downward direction and to avoid faulty analysis results, ethanol is removed first by centrifuging the vessel. The ethanol thus escapes from the membrane and can be removed in a downward direction. Subsequently, the membrane is sufficiently free from ethanol. Ethanol can alternatively be removed from the membrane by means of vacuum and the supply of heat.
Elution is carried out in order to dissolve the nucleic acid from the membrane again. As a rule, this is done using water or a weakly saline, aqueous, pH-stabilized solution. Subsequent to the elution, the nucleic acid can be removed from the vessel in a downward direction through the membrane.
What is problematic in this prior art is that the vessel or a device comprising this vessel has to be opened time and again, as can be seen from the publication “QlAamp® DNA Mini and Blood Mini Handbook, Second Edition, November 2007 (see http://www1.qiagen.com/HB/QlAampDNAMiniAndDNABloodMiniKit_EN). Thus, the sample can escape to the outside. There is a risk of an unwanted contamination.
However, the disruption of a biological sample is carried out in a second vessel better suited to this purpose, in order thus to be able to carry out a disruption particularly effectively. When the biological sample has been disrupted in a second vessel, then it is subsequently filled into the vessel comprising the filter element, that is, a column, and processed. The risk of contamination is increased further by the transfer.
There are various publications regarding the topic of the processing of biological materials. For example, U.S. Pat. No. 6,060,022 discloses an automated system for sample processing comprising an automated centrifuging apparatus. U.S. Pat. No. 5,166,889 describes a collecting system for blood in which a plurality of collecting vessels is positioned in a carrier wheel for direct access. U.S. 2004/0002415 describes an automated centrifuging system for automatically centrifuging liquids containing biological material, such as nucleic acid, in a general centrifuge. WO 2005/019836 describes an apparatus for processing liquid samples. WO 00138046 describes an automated device for charging a centrifuge, in which columns are provided to the centrifuge via an automated routing system. EP 122772 describes a chemical manipulator for use with reaction vessels. GB 2 235 639 describes a centrifuge with a protective casing surrounding the rotating bowl shell. According to WO 2006/042838 A1, a single-use cartridge is proposed which comprises a system of micro-channels and micro-cavities for a predetermined process after the sample has been accommodated. An apparatus for processing biological material is known from DE 10 2006 027 680 A1. DE 102005053463 A1 discloses a reaction unit comprising a bottom part and a top part, for pipetting liquids, wherein the bottom part consists of a reaction cavity with a permeable filter mesh insert and the top part constitutes a reaction cavity that can be affixed to the bottom part and contains a recess or sleeve for accommodating a magnet. Nucleic acids are bound to magnetic particles. Devices for automatically processing known from the prior art often are disadvantageous in that a technically complex apparatus has to be used. The device known from U.S. 2006/0281094 is an example for a technically complex apparatus. Such devices are relatively expensive and require a relatively large amount of space. It is not possible to integrate such a complex and large apparatus into microfluidic systems, such as those known from WO 2006/042838 A1.
Microfluidic systems are advantageous for the integration of complex processes in closed systems (lysis, sample preparation, amplification, detection). This demand for closed systems arises in the field of diagnostics, in which cross contamination has to be avoided. Cross contaminations can be particularly serious if very sensitive detection methods (e.g. PCR—polymerase chain reaction, that is, a method in which minute amounts of a DNA portion can be multiplied in a chain reaction) are used. Moreover, operating errors are avoided by the integration of the procedures.
A microfluidic system for handling and/or detecting particles such as cells or glass beads is known from document U.S. 2004/0229349. According to the fifth example disclosed in this document, a microfluidic system comprises a feeding channel and a waste channel, which are interconnected via filter channels. Particles get into the filter channels via the feeding channel. The particles, for example erythrocytes, are retained by filters located in the filter channels. However, liquids can still get into the waste channel from the feeding channel via the filter channels. By reversing the direction of flow of the liquid through the filter channels, as well as after the suitable actuation of valves, the particles are finally transported out of the filter channels into an analysis zone. It is not known from U.S. 2004/0229349 to feed buffers used for processing into the vessel with a biological sample contained therein via an inlet or outlet through a filter of a vessel.
In order to increase sensitivity alternatively or additionally to PCR, large sample volumes can be prepared. The preparation of large volumes, however, is contradictory to the demand for microfluidic systems for automatic lysis, processing and/or analysis of biological samples. There is therefore a demand for solutions which permit preparing a large sample volume by means of, for example, filtration, and to make the isolated nucleic acids available in a small volume to a microfluidic system via a microfluidic interface. Such a microfluidic interface for membrane-based preparation methods is not known. As a rule, this problem is solved in integrated micro-systems by means of bead-based purification processes.
An extraction device with a filter unit for automatically carrying out the processing (binding-washing-eluting) of nucleic acids is known from EP 1382675 A1 . In this case, a sample that has been previously lysed is put on the filter unit by the user. Passing the sample through the filter membrane can be carried out by means of a vacuum. Subsequent washing solutions are automatically added and sucked over the membrane. The elution step takes place in the same way after an elution buffer has been added. For the following reasons, this device is not suitable for connection to a microfluidic system if the vessel volume is supposed to be large.
The “bind-wash-elute” process is always carried out for sample preparation with filter-based spin columns. In this case, the liquids are put on the membrane only from the top—as is the case, for example, in the device known from EP 1382675 A1—and drained off at the bottom on the outlet (by centrifugation or vacuum). If the volume of the vessel or column is too large to be able to accommodate large quantities of liquid, the latter is expediently configured such that large volumes can easily be fed to it from the top. For elution in small volumes, however, the liquid must be pipetted exactly onto the center of the membrane in order to accomplish an efficient elution. Thus, feeding small quantities of liquid from the top cannot be carried out for a connection to microfluidic systems, or only with difficulties, and is not advantageous in any case.